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Study of molecular and cellular pathogenicity mechanisms of Arcobacter species

Publication . Ferreira, Susana Margarida Paraíso; Domingues, Fernanda da Conceição; Queiroz, João António de Sampaio Rodrigues; Oleastro, Mónica Alexandra de Sousa

Arcobacter is a genus of Gram-negative, spiral-shaped bacteria in the Epsilonproteobacteria class. It was first proposed in 1991 and was included in the Campylobacteraceae family together with the genera Campylobacter and Sulfurospirillum. There are currently 18 species described, among which A. butzleri, A. cryaerophilus and A. skirrowii are known to be human and animal pathogens. Arcobacter genus shows an unusually wide range of habitats, having been isolated from food, water, food processing and handling facilities, diverse environmental, animal and human samples. In fact, consumption of Arcobacter-contaminated food or water is regarded as the most probable cause of infection by this bacterium. Therefore it is important to evaluate its distribution in food or food processing environment, together with the assessment of its genetic variability and other phenotypic and genotypic features associated with virulence potential, to be able to understand the persistence mechanism of Arcobacter in the food chain. Thus, this study intend to evaluate the presence, genetic diversity, and virulence characteristics (namely antibiotic resistance, biofilm-forming capacity and detection of putative virulence genes) of A. butzleri strains isolated from poultry and from the environment of a Portuguese slaughterhouse. A. butzleri isolates revealed a high genetic diversity, with all isolates showing to be susceptible to gentamicin, in contrast to 55.8% that were resistant to ciprofloxacin. The latter resistance was associated with the presence of a cytosine to thymine transition in the quinolone resistance determining region of the gyrA gene. Among selected isolates, 72.2 % presented biofilm-forming ability and in all strains putative virulence genes were detected. These results highlight the relevance of A. butzleri relevance as food-borne pathogen. Taking into account that Arcobacter is potentially transmitted through contaminated food and is resistant to common antimicrobials, it is important to develop alternative control strategies that could be both effective and safe for human consumption. Therefore, the antimicrobial properties of resveratrol against A. butzleri and A. cryaerophilus were studied. Resveratrol exhibited a bacteriostatic or bactericidal activity dependent on cellular growth phase and resveratrol concentration, leading to both DNA content and metabolic activity reduction on Arcobacter cells. Resveratrol also showed the ability to act as an efflux pump inhibitor, and to induce cellular damage. Thus, resveratrol showed anti-Arcobacter activity, with the results obtained suggesting that this compound inhibits this microorganism through different pathways, which together with resveratrol beneficial properties described for human health may encourage its use as a food preservative. Some of the species of Arcobacter genus have been associated with gastrointestinal disease in humans, however there were a lack of studies evaluating its prevalence in Portugal, with the same happening for non-Campylobacter jejuni/coli species. Therefore, the frequency of Arcobacter and Campylobacter species in faeces from patients with diarrhoea in Portugal was assessed using a molecular approach. Concerning, Arcobacter and Campylobacter prevalence and distribution, 298 diarrhoeal samples from Portuguese patients were analysed, 1.3% of the samples were positive for A. butzleri and 0.3 % for A. cryaerophilus. Campylobacter species were found in 31.9 % of diarrhoeic faeces samples, with C. jejuni and C. concisus being the most prevalent species of this genus (13.7 % and 8.0 %, respectively). In this cohort of samples, A. butzleri was the fourth most frequent species. These results evidence the importance of Arcobacter and Campylobacter species as aetiological agents of acute gastroenteritis among Portuguese patients, affecting particularly the paediatric age group. Although A. butzleri has been implicated in human diseases, much of its pathogenesis and virulence factors remain unclear. Thus, A. butzleri virulence potential was also investigated, through the characterization of genotypic and pathogenic properties of human and non- human isolates. The isolates showed to be susceptible to tetracyclines and aminoglycosides, however displaying high resistance to quinolones. A. butzleri demonstrated a weak haemolytic activity and the ability to form biofilms in polystyrene surfaces. Adhesion levels similar to Salmonella Typhimurium were found for A. butzleri on Caco-2 cells, with pre- existing inflammation showing no significant effect on its adherence ability, yet invasion ability showed to vary among the isolates. A. butzleri was able of intracellular survival in Caco-2 cells and to induce a significant up-regulation of interleukin-8 secretion, as well as to promote structural cell disturbance. These data brings new insights to A. butzleri virulence and highlights its pathogenic potential. Overall in this work, the prevalence of Arcobacter species both in human and food-related samples was evaluated, contributing to understanding the epidemiology of Arcobacter in Portugal. The survival and persistence of this organism in the environment was highlighted due to its ability to form biofilms. Its relevance as a human pathogen was underlined by the resistance to antimicrobials, the presence of several putative virulence genes, along with its adherence, invasion, intracellular survival abilities and induction of proinflammatory cytokine secretion in intestinal epithelial cells. Finally, resveratrol was tested as an alternative to control the growth of Arcobacter. This work provided new insights on the epidemiology and pathogenicity of Arcobacter, and also identified a natural compound with anti-Arcobacter activity, which may contribute for future developments of new control approaches.

2014-05Doctoral thesis

Open access

Evaluation of the prebiotic potential of arabinoxylans from brewer's spent grain

Publication . Reis, Sofia F; Gullón Estévez, Beatriz; Gullón, Patricia; Ferreira, Susana; Baptista, Cláudio; Alonso, José L; Domingues, F.C.; Abu-Ghannam, Nissreen

Arabinoxylans (AX) consumption has been related to the treatment and prevention of cardiovascular diseases, type II diabetes, colorectal cancer and obesity. The beneficial health effects are conferred through gut microbiota modulation, and therefore, they have been proposed as potential slowly fermentable prebiotic candidates. As the mechanisms are not yet well understood, the prebiotic potential of AX from brewer's spent grain (BSG) has been investigated. Two types of AX from BSG (AX1 and AX2) of different length and branching averages were fermented with human faecal inocula and compared to fermented cultures containing a commercial prebiotic (fructooligosaccharide (FOS)) and cultures with no added carbohydrate (control). Results demonstrated that the AX were extensively metabolised after 48 h of fermentation. The pH decreased along fermentation and the lowest value was achieved in AX1 cultures. The production of short chain fatty acids (SCFA) was higher in AX cultures than in cultures containing FOS and controls, with AX1 presenting the highest concentrations. The stimulatory effect of beneficial bacteria was higher in AX cultures, and AX2 presented the highest positive effect. Prebiotic potential of AX from BSG was confirmed by the production of SCFA and the modulation of gut microbiota, especially by the high increase in bifidobacteria populations.

2014-11Journal article

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Molecular diagnosis of Arcobacter and Campylobacter in diarrhoeal samples among Portuguese patients

Publication . Ferreira, Susana; Júlio, Cláudia; Queiroz, João; Domingues, F.C.; Oleastro, Mónica

The present study was conducted to investigate the prevalence and diversity of Arcobacter and Campylobacter spp. in 298 stool samples of patients with diarrhoea, collected from 22 Portuguese hospitals, between September and November 2012. Detection of Arcobacter and Campylobacter spp. was performed using molecular-based detection techniques, such as real-time fluorescence resonance energy transfer PCR, species-specific PCR, and sequencing of amplified PCR products. Overall, 1.3% of the samples were positive for Arcobacter butzleri and 0.3% for Arcobacter cryaerophilus. Campylobacter spp. were found in 31.9% of diarrhoeic faeces. Campylobacter jejuni and Campylobacter concisus were the most prevalent species (13.7% and 8.0%, respectively). The prevalence of Arcobacter and Campylobacter spp. was significantly different between children and adults (39.7% versus 22.8%, P = 0.003). We underline the high prevalence of these pathogens in diarrhoeal samples among Portuguese patients, with particular relevance in the paediatric age group.

2014Journal article

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Genotypic and phenotypic features of Arcobacter butzleri pathogenicity

Publication . Ferreira, Susana; Queiroz, João; Oleastro, Mónica; Domingues, F.C.

Even though Arcobacter butzleri has been implicated in some human disease as diarrhoea and bacteraemia, much of its pathogenesis and virulence factors remain unclear. In this work we have compared pathogenic and genotypic properties of six A. butzleri isolates from human and non-human sources. The tested isolates showed to be susceptible to tetracyclines and aminoglycosides, however non-human isolates were all resistant to quinolones. The ability to form biofilms was variable among the tested strains, and all of them showed a weak haemolytic activity. The presence of nine putative virulence genes was determined, with cadF, ciaB, cj1349, mviN, pldA, tlyA being detected in all strains, while irgA (3/6), hecA (5/6), hecB (4/6) were detected only in some strains. High levels of adhesion were observed for A. butzleri on Caco-2 cells, with pre-existing inflammation showing no significant effect on the adherence ability; yet variable levels of invasion were observed. A. butzleri isolates were able to survive intracellularly in Caco-2 cells and to induce a significant up-regulation of interleukin-8 secretion and structural cell rearrangements. These data brings new insights on A. butzleri virulence and highlights its pathogenic potential.

2014Journal article

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Insights in the pathogenesis and resistance ofArcobacter: A review

Publication . Ferreira, Susana; Queiroz, João; Oleastro, Mónica; Domingues, F.C.

Arcobacter genus currently comprises 18 recognized species, among which Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii have been associated with human and animal disease. Although these organisms, with special emphasis A. butzleri, are emerging as clinical pathogens, several aspects of their epidemiology and virulence are only starting to be clarified. In vitro human and animal cell culture assays have been used to show that several Arcobacter species can adhere to and invade eukaryotic cells, induce an immune response and produce toxins that damage host cells. In addition, data from genome sequencing highlighted several potential markers that may be helpful candidates for the study and understanding of these mechanisms; however, more work is necessary to clarify the molecular mechanisms involved in Arcobacter virulence. Arcobacter can be considered a relatively robust organism showing to be able to survive in adverse conditions, as the ones imposed by food processing and storage. Moreover, these bacteria have shown increased antibiotic resistance, along with high multidrug resistance. In this review, we seek to update the state-of-the-art concerning Arcobacter distribution, its interaction with the host, the trends of antibiotic resistance, its ability to survive, and finally the use of natural antimicrobials for control of Arcobacter.

2016Journal article

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